1. Field of the Invention
The present invention relates to mass spectrometry systems. More particularly, it relates to mass spectrometry systems that are useful for the analysis of complex mixtures of molecules, including large organic molecules such as proteins or peptides, environmental pollutants, pharmaceuticals and their metabolites, and petrochemical compounds, to methods of analysis used therein, and to a computer program product having computer code embodied therein for causing a computer, or a computer and a mass spectrometer in combination, to affect such analysis. Such mass spectrometry systems may also include ion mobility spectrometers which typically operates at ambient pressure without a vacuum pump typical of a mass spectrometer. The data acquired from IMS is typically called a plasmagram.
2. Background Art
Typical mass spectral data, acquired from either individual MS scans or averaged scans, contain rich information about the sample under study, including the molecular ions, fragment ions, adducts, the electrical charges, the concentrations, and the impurities (or co-eluting interferences from LC/MS or GC/MS experiments). It is highly desirable to determine from the mass spectral data the following information for small as well as large molecules:                The purity of a particular mass spectral peak.        If a mass spectral peak is deemed impure, the number of possible components contained in the peak and, furthermore, the elemental compositions related to each component.        
Large biomolecules such as proteins or peptides, under electro-spray ionization (ESI), typically become multiply charged and become observable at low m/z ranges on a quadrupole mass spectrometer. Since the isotope distribution for these large molecules is relatively wide, covering quite a few mass units, the observed peak width for a particular molecule at a given charge is contributed to by both the isotope distribution and the intrinsic mass spectral peak width. Knowing the mass spectral peak width and determining the charge state for an observed mass spectral peak allows one to estimate the mass of the original large biomolecule, an important step in identifying key proteins or peptides for proteomics applications.
This task of peak purity determination and/or charge state determination has been quite a challenge due to the lack of dependable peak shape information through conventional mass spectral data processing, requiring user knowledge and human intuition for peak purity analysis and multiple observable peaks of consecutively varying charges for charge state determination.
Mass spectrometers, especially ion trap types of mass spectrometers, generally suffer from space charge effect, where mass spectral shift and possibly peak shape change occurs, thus limiting the mass accuracy achievable and usefulness for related applications, including mass spectral purity detection, charge determination, elemental composition determination, etc. While hardware solutions such as linear ion traps, 3D traps, and ion traps with larger internal volumes have been proposed, a software solution is preferred as it can benefit MS users of existing MS systems and further enhance the MS capabilities of newer designs.
Liquid chromatography interfaced with (tandem) mass spectrometry (LC/MS or LC/MS/MS) has been widely utilized for obtaining structural information of molecules such as the sequence of proteins and metabolic pathways of pharmaceuticals. To study a drug and its metabolites, the drug is typically injected into an animal model and biological fluids are taken from the animal model as samples for subsequent sample preparation, such as extraction and LC/MS analysis. The drug and its metabolites are separated in time and then detected with mass spectrometry. To search for a particular molecule, either the drug itself or its possible metabolites, the user would go through a post-analysis process to extract ion chromatograms in a limited m/z window. For verapamil (C27H39N2O4+, monoisotopic mass 455.2910 Da), for example, the drug itself can be seen in an extracted ion chromatogram in the m/z range of 454.8 and 455.8. This approach suffers from several drawbacks:                1. On conventional unit mass resolution systems, the mass spectral centroiding process can rarely provide better than 0.1 Da in mass accuracy, necessitating ion integration in a large mass window such as +/−0.5 Da.        2. While such large mass window has the potential advantages of getting more ions integrated with better signal-to-noise, it at the same time opens up the window for unwanted ions from background and matrices, complicating the extracted ion chromatogram and its interpretation.        3. Even on higher resolution MS systems where one could afford to narrow the integration window due to the narrower peak width and higher mass accuracy achievable, such ion extraction process is prone to errors caused by including the isotope ions of other ions. In the above example, the M+1 isotope cluster from another ion at 454.291 will show up in the m/z window of verapamil and be included as the ion of interest.        
Due to these complications, LC/MS data processing and interpretation typically takes longer than the LC/MS experiment itself, in spite of an apparently complicated multi-step process involved in acquiring the data through sample preparation, LC separation and MS analysis. The presence of biological matrices such as bile, feces and urine further complicates the analysis due to the many background ions these matrices generate. There are currently two approaches to address the issue of complex matrices:                Use a higher resolution system such as qTOF where the higher resolution and better mass accuracy can lead to better separation and differentiation between the ions of interest and those coming from the background matrices.        Perform further MS analysis through MS/MS experiments that offer a variety of structurally specific information to facilitate identification of metabolites and proteins/peptides in the presence of biological matrices.        
MS/MS experiments in general require two mass analyzers and a collision cell filled with collision gas such as Argon. The combination of scan functions from each of the two mass analyzers results in three different MS/MS modes. One aspect of the design of MS/MS experiments is the selection of the three MS/MS operational modes currently available for qualitative analysis:                1. Product ion scan. This is the most commonly used MS/MS experiment for structural elucidation. Its experimental process includes the selection of precursor ions by one mass analyzer, collision-induced dissociation (CID) of the precursor ions in the collision cell, and scanning of the fragment ions by another mass analyzer. This scan mode can be performed in all the instruments that have two mass analyzers such as triple stage quadrupole (TSQ), ion trap, quadrupole/time of flight (qTOF), Fourier Transform mass spectrometer (FTMS), and Time-Of-Flight/Time-Of-Flight (TOF/TOF).        2. Neutral loss scan. This scanning function is currently available only in a TSQ instrument. By scanning two quadrupoles at the same time with a mass offset equal to the mass of lost neutrals during CID, this method detects only those ions that lost a specific functional group and is useful for target analysis.        3. Precursor ion scan. This is another scanning function available only in a TSQ instrument for target analysis based on a specific fragment ion. With the first mass analyzer scanning in a certain mass range, the second mass analyzer is set to detect the specific ions generated by CID.        
Another aspect of the design of MS/MS experiments is how to execute the MS/MS experiments during an LC separation. Early LC/MS/MS data acquisition required a pre-run to determine the retention time and the m/z value of the precursor ions of interest to define the time window for MS/MS on the precursor ions and to set up MS/MS conditions such as mass scan range and collision energy, respectively. This inefficient and hardly automated data collection procedure quickly prompted the development of data dependent acquisition or information dependent acquisition. Data dependent acquisition is an intelligent way to perform MS/MS on-the-fly without the knowledge of retention time and the m/z value of the precursor ions. It starts with a survey scan (usually a full MS scan), to calculate the intensities of the most abundant ions and their signal-to-noise ratios for decision-making by the data acquisition system. If ion intensities and/or signal-to-noise ratios exceed a pre-defined threshold, the acquisition will be triggered to switch from full MS scan to product ion scan. After the MS/MS, data collection continues to perform a full MS scan until the intensities and/or signal-to-noise ratios are above the threshold to trigger the MS/MS again. This cycle from full MS to MS/MS usually repeats itself through an entire LC run.
Data dependent acquisition can also use the neutral loss scan or the precursor scan as the survey scan to achieve more specificity. For example, when the ions losing particular neutrals during CID are determined to be of interest, the neutral loss scan is employed to find those ions. As soon as a signal is detected by the neutral loss scan with sufficient intensity and/or signal-to-noise ratio (above a preset threshold), the data acquisition will switch from the neutral loss scan to product ion scan. This powerful combination provides high specificity by neutral loss or precursor scan and detailed structural information by product ion scan, all in one experiment.
Over the past 10 years or so, data dependent acquisition has increasingly gained great popularity for high throughput applications such as metabolic profiling and proteomics applications. However, due to the highly non-specific criteria, namely, the ion intensities and signal to noise ratios, used to trigger MS/MS experiments in the data dependent acquisition, MS/MS spectra are generated not only from the ions of interest such as metabolites in metabolic profiling applications, but also from the ions of the matrix or the background. This poses a challenging problem for automated post-acquisition data processing. Moreover, when the ions of interest co-elute with, and are less abundant than the background or matrix ions, the ions of interest will be skipped while the background or matrix ions are selected for the product ion scan.